JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY VOL. 70, NO. 22, 2017 ª 2017 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION ISSN 0735-1097/$36.00 PUBLISHED BY ELSEVIER https://doi.org/10.1016/j.jacc.2017.10.002 THE PRESENT AND FUTURE STATE-OF-THE-ART REVIEW Genome Editing The Recent History and Perspective in Cardiovascular Diseases Kiran Musunuru, MD, PHD, MPH ABSTRACT The genome-editing field has advanced to a remarkable degree in the last 5 years, culminating in the successful correction of a cardiomyopathy gene mutation in viable human embryos. In this review, the author discusses the basic principles of genome editing, recent advances in clustered regularly interspaced short palindromic repeats and clustered regularly interspaced short palindromic repeats–associated 9 technology, the impact on cardiovascular basic science research, possible therapeutic applications in patients with cardiovascular diseases, and finally the implications of potential clinical uses of human germline genome editing. (J Am Coll Cardiol 2017;70:2808–21) © 2017 by the American College of Cardiology Foundation. n adult man has been diagnosed with famil- carry to term a child who would be free of the A ial hypertrophic cardiomyopathy, with father’sdisease. severe enough disease to warrant the use Although this vignette might have the feel of of an implantable cardioverter-defibrillator and anti- science fiction, in fact this very sequence of events arrhythmic medications. Upon genetic testing, he is has already occurred in real life (1), published in a found to be heterozygous for a 4-bp deletion in the report in August 2017 and widely announced in the MYBPC3 (myosin-binding protein C, cardiac type) press. In this case, there was never any intent for the gene. In light of this finding, each of his children embryos to be carried to term, but the embryos were would have a 50% chance of inheriting the MYBPC3 viable and in principle could have given rise to live mutation and being at risk for severe hypertrophic people. This highlights the dramatic progress that has cardiomyopathy. To avoid this possibility, the taken place in the field of genome editing. How did man’s sperm are used for in vitro fertilization of we get to this point? oocytes. At the same time that the sperm are injected into the oocytes, clustered regularly inter- PRIMER ON GENOME EDITING spaced short palindromic repeats (CRISPR)–CRISPR- associated 9 (Cas9) and a synthetic deoxyribonucleic It is important to recognize that genome editing did acid (DNA) molecule containing the correct MYBPC3 not suddenly emerge out of a vacuum but built on sequence are also injected with the intent of cleanly decades of work by numerous investigators seeking to correcting the mutation carried by the sperm. This improve the ability to target specific alterations into procedure results in many embryos in which the specific genes within the genomes of cells, whether in correction has successfully occurred, with no effects model organisms such as mice or in human cells. on the rest of the genome. The embryos are ready Traditional gene targeting, such as that used in mouse for transfer into the womb of a mother, who would embryonic stem cells to make “knockout” mice, is Listen to this manuscript’s audio summary by JACC Editor-in-Chief Dr. Valentin Fuster. From the Cardiovascular Institute, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; and the Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. This work was partially supported by National Institutes of Health grants R01-HL118744, R01-GM104464, R01-DK099571, and R01-HL126875, and the American Heart Association. Dr. Musunuru has reported that he has no relationships relevant to the contents of this paper to disclose. Manuscript received August 20, 2017; revised manuscript received October 2, 2017, accepted October 3, 2017. JACC VOL. 70, NO. 22, 2017 Musunuru 2809 DECEMBER 5, 2017:2808– 21 Genome Editing in CVD technically challenging and relies on the process of produce 2 double-strand breaks on the same ABBREVIATIONS homologous recombination (2). A custom-made piece chromosome, the portion of the DNA mole- AND ACRONYMS of DNA is introduced into cells and serves as the cule between the breaks might be irrevocably AAV = adeno-associated virus template for homologous recombination. For the lost if the far free ends are rejoined. The Cas9 = clustered regularly custom-made template to work, it must have se- consequence could be deletion of part of a interspaced short palindromic quences that match the sequences around the target gene, an entire gene, or a chromosomal re- repeats–associated 9 — site in the genome regions of homology, also termed gion with multiple genes. CHD = coronary heart disease homology arms. A desired alteration, whether a single The second way in which a cell can repair a CRISPR = clustered regularly nucleotide change or a longer DNA sequence to be double-strand break is homology-directed interspaced short palindromic inserted, is placed between the homology arms in the repair (HDR). Unlike NHEJ, HDR is normally repeats template. Homologous recombination causes limited to proliferating cells that have CRISPRa = clustered regularly interspaced short palindromic “crossing over” to occur between the matching ho- doubled their chromosomes and thus have repeats activation mology arms in the genome and in the introduced duplicate chromatids on each chromosome CRISPRi = clustered regularly template and stably copy the alteration into the (i.e., are in S phase or G2 phase). Akin to ho- interspaced short palindromic genome. Spontaneous homologous recombination mologous recombination, HDR requires a repeats interference with a custom-made template occurs at a very low repair template with homology to the DNA DNA = deoxyribonucleic acid frequency—on the order of a 1-in-a-million event. sequences flanking the double-strand break. HDR = homology-directed While there are methods to enrich for cells in which Ordinarily the repair template is a duplicate repair the desired event has occurred and discard the other chromatid(whichhastheidenticalsequence) hPSC = human pluripotent cells(whichhasmadeitpossibletogenerategeneti- or a matching chromosome (e.g., the pater- stem cell cally modified cells and animals in the laboratory nally inherited chromosome that is paired iPSC = induced pluripotent stem cell setting), the frequency of recombination is far too low with the maternally inherited chromosome— NHEJ = nonhomologous end to be useful if the goal is to produce a therapeutic ef- perhaps not identical in sequence, but similar joining fect in cells in a live human being. enough to provide homology) (5).Asynthetic, PAM = protospacer-adjacent Genome editing takes advantage of tools that pro- custom-made DNA template with a desired motif fl duce double-strand DNA breaks at desired locations in alteration anked by homology arms, when RNA = ribonucleic acid the genome. The double-strand break activates the introduced into a cell, can instead be used by TALEN = transcription cellular DNA repair machinery and, in doing so, can HDR and result in stable copying of the activator-like effector nuclease fi improve the ef ciency of altering the genome by or- alteration into the genome (6). ZFN = zinc-finger nuclease ders of magnitude. Instead of being 1-in-a-million Whereas NHEJ can have unpredictable events, alteration of the genome can routinely occur consequences—indels of varied sizes occur, sometimes with higher than 1-in-10 frequency. This dramatic as large as kilobases, which can affect genes beyond improvement in efficiency has for the first time made just the target gene—HDR is a much more precise repair it feasible to undertake a “rewriting” of the human process that makes it suitable for correcting disease genome for therapeutic purposes. mutations. However, HDR has 3 principal disadvan- Genome editing that is instigated by double-strand tages compared with NHEJ. First, HDR-mediated breaks can achieve several types of changes, tied to editing generally occurs with less efficiency than the 2 major ways in which the cell repairs double- does NHEJ-mediated editing in proliferating cells, due strand breaks (Figure 1) (3).Innonhomologousend to HDR being limited to only part of the cell cycle. joining (NHEJ), the DNA molecule’sfreeendscreated Although exceptions have been observed in vitro in by the double-strand break are rejoined (4).NHEJis transformed cells (in which DNA repair mechanisms the default repair pathway in the sense that it are often dysregulated) (7), and certain chemicals have operates in all types of cells at all times. NHEJ is a been shown to inhibit NHEJ or enhance HDR in less-than-perfect repair process that occasionally cultured cells in vitro (8–10), in vivo studies appear to results in the semirandom insertion or deletion of confirm this rule (11,12). Second, HDR does not DNA base pairs, termed “indels.” Indels introduced normally occur at all in nonproliferating cells, a into the coding sequence of a gene will either repre- substantial limitation with respect to postnatal sent frameshift mutations or in-frame insertions or cardiomyocytes and other cell types relevant to deletions; the former will usually result in scrambling cardiovascular disease. Third, HDR-mediated of part of the amino acid sequence and premature editing requires a custom-made repair template, and truncation of the protein product of the gene, and the